cAMP affects 5-HT-induced Ca2+ signaling
Threshold concentrations of 5-HT (1–3 nM) induced intracellular Ca2+ oscillations, whereas saturating 5-HT concentrations (> 30 nM) produced biphasic Ca2+ responses that consisted of an initial transient followed by a plateau of elevated [Ca2+]i (Figs. 1A, B, and [26,27]).
To test whether these two types of response patterns were affected by
cAMP, we increased the intracellular cAMP by bath application of 10 mM
cAMP, 100 μM IBMX, or 100 μM forskolin. These substances/concentrations had no effect on resting [Ca2+]i . As shown in Fig. 1A, 3 nM 5-HT induced intracellular Ca2+ oscillations,
as described previously. Application of forskolin to the bath in the
continuous presence of 3 nM 5-HT converted the oscillatory [Ca2+]i changes
into a sustained increase (n = 8). Treatment with cAMP or IBMX had the
same effect as forskolin at all tested preparations (cAMP, n = 7; IBMX,
n = 5). Forskolin did not affect the sustained Ca2+ elevation produced by 30 nM 5-HT (Fig. 1B), a concentration that saturates the rate of fluid secretion .
Figure 1. Forskolin augments [Ca2+]i changes induced by low 5-HT concentrations in Calliphora salivary gland cells. (A) Stimulation with 3 nM 5-HT produces intracellular Ca2+ oscillations. Application of 100 μM forskolin converts oscillatory [Ca2+]i changes
into a sustained increase (n = 8). (B) Stimulation of the gland with 30
nM 5-HT, a concentration that saturates fluid secretion, produces a
biphasic Ca2+ response consisting of an initial transient followed by a plateau of elevated [Ca2+]i. The sustained phase of elevated [Ca2+]i is not effected by forskolin (n = 4). (C) Application of a threshold concentration of 5-HT (1 nM) in Ca2+-free PS (0-Ca, 2 mM EGTA) increases [Ca2+]i just measurably without triggering Ca2+ spikes. Additional application of 100 μM forskolin induces a transient Ca2+ elevation, showing that forskolin augments 5-HT-induced Ca2+ release, not Ca2+ entry (n = 8).
To determine whether the extra Ca2+ increase produced by forskolin at low 5-HT concentrations was attributable to Ca2+ influx
from the extracellular space, we stimulated glands with a sub-threshold
concentration of 5-HT (in order to prevent fast Ca2+ store depletion ) and applied forskolin in Ca2+-free PS (no added Ca2+, 2 mM EGTA). As seen in Fig. 1C, 1 nM 5-HT was below the concentration that induced marked Ca2+ oscillations (in Ca2+-containing PS), but application of 100 μM forskolin stimulated a transient Ca2+ elevation even in the absence of extracellular Ca2+. Taken together, these results suggested that cAMP did not induce Ca2+ influx but rather augmented Ca2+ release from the ER produced by low 5-HT concentrations.
cAMP augments InsP3-induced Ca2+ release from the ER
Theoretically, there are two mechanisms for the release of Ca2+ from the ER: the InsP3R and the ryanodine receptor Ca2+ channel (RyR). Blowfly salivary glands, however, seem to lack RyR , leaving only the InsP3R as potential target for the cAMP pathway in order to enhance Ca2+ release.
To examine directly whether cAMP augmented InsP3-induced Ca2+ release we studied Ca2+ release from the ER by intraluminal Ca2+ measurements with the low-affinity Ca2+-indicator dye Mag-fura-2. This dye accumulates within the ER and after β-escin
permeabilization of the plasma membrane in an artificial "intracellular
medium" (ICM) and loss of cytosolic dye, it monitors intraluminal Ca2+ ([Ca2+]L) [32,35,36]. Figures 2A and 2B show two representative original recordings of intraluminal Ca2+ measurements.
In order to facilitate the quantitative evaluation of this type of
measurements, we converted Mag-fura-2 fluorescence ratios into a
percentage scale, with 0% Ca2+ release representing the intraluminal Mag-fura-2 ratio at time zero of the recording, and 100% Ca2+ release representing the fluorescence ratio after the loss of intraluminal Ca2+ following ionomycin application.
Figure 2. cAMP augments InsP3-induced Ca2+ release from β-escin permeabilized cells, as shown by intraluminal Ca2+ measurements with Mag-Fura-2. (A) cAMP does not induce Ca2+ release from the ER (n = 4). (B) Application of 5 μM InsP3 induces Ca2+ release from the ER and is augmented by 100 μM cAMP. (C, D) Ca2+ release induced by 5 μM InsP3 is neither enhanced by application of fresh InsP3 solution (C) nor by mock stimulation with Rp-cAMPS (D). (E) Quantification of the cAMP-dependent augmentation of Ca2+ release induced by 5 μM InsP3 from experiments as shown in B. 0% Ca2+ release is the intraluminal Mag-fura-2 ratio at time zero of the recording; 100% Ca2+ release is the fluorescence ratio after complete loss of intraluminal Ca2+ following ionomycin application. A sigmoidal dose-response curve fitted to mean values (R2 = 0.4) of the InsP3(+cAMP)-induced Ca2+ release gives an EC50, cAMP of 2.6 μM. (F) Dose-response relationship for InsP3-induced Ca2+ release in the presence (triangles) and absence (squares) of 100 μM cAMP. The leftward shift of the dose-response relationship indicates sensitization of InsP3-induced Ca2+ release for InsP3 by cAMP. (E, F) The number of measurements for every data point is given in brackets. Means ± S.D.
Application of 100 μM cAMP to the permeabilized gland tubules did not induce Ca2+ release from the ER, whereas the Ca2+-ionophore ionomycin led to a dramatic loss in intraluminal Ca2+ (Fig. 2A). Treatment with 5 μM InsP3, on the other hand, caused a partial Ca2+ release, and the subsequent addition of 100 μM cAMP resulted in a further Ca2+ release (Fig. 2B), indicating that cAMP had augmented InsP3-induced Ca2+ release. In order to obtain the dose-response relationship for the effect of cAMP on InsP3-induced Ca2+ release, the cAMP concentration was systematically varied, and Ca2+ release (%) (Fig. 2E, squares) was measured after cAMP addition to ICM containing 5 μM InsP3. The sigmoidal dose-response curve fitted to the mean values of the InsP3(+cAMP)-induced Ca2+ release gave a mean half maximal cAMP concentration (EC50) of 2.5 μM (Fig. 2E).
In order to exclude that the augmentation of InsP3-induced Ca2+ release was not simply the result of the addition of fresh InsP3(+cAMP)-containing ICM, we superfused several preparations with InsP3(no cAMP)-containing ICM twice. A second InsP3 application never increased Ca2+ release induced by a prior InsP3 application (Fig. 2C; n = 5). Moreover, mock stimulation with 10 μM (n = 5) or 100 μM (n = 5) 8-Br-Rp-cAMPS (a competitive antagonist of cAMP binding to PKA) had no significant effect on the InsP3-induced Ca2+ release (Fig. 2D displays a representative original recording with 10 μM 8-Br-Rp-cAMP).
To determine whether cAMP increased the affinity of the InsP3R for InsP3, we examined Ca2+ release induced by increasing InsP3-concentrations in the absence (Fig. 2F, squares) and presence of 100 μM cAMP (Fig. 2F, triangles). The two resulting dose-response curves indicated that cAMP increased the affinity of the InsP3R for InsP3, because cAMP shifted the dose-response curve to lower InsP3 concentrations by about one order of magnitude.
Is the cAMP-dependent augmentation of InsP3-induced Ca2+ release mediated by PKA or EPAC?
The effect of cAMP on InsP3-induced Ca2+ release could be mediated by either PKA or Epac. Both target proteins are expressed in blowfly salivary glands .
To distinguish between these possibilities, cAMP-analogs that activate
either PKA or Epac or both downstream effectors were used instead of
cAMP . These cAMP analogs were applied at concentrations of 10 μM and 100 μM. One problem in the quantitative evaluation of these experiments was, that the Mag-fura-2 fluorescence ratio in the β-escin-permeabilized preparations continuously declined as Ca2+ leaked out of the ER (see, for example, Figs. 2A, B; 3A, C, D),
and this decline in fluorescence ratio varied between preparations.
Therefore, we did not measure and compare the magnitude of Ca2+ release
from the ER (as above), but rather its rate as measured by the decline
in the Mag-fura-2 fluorescence ratio per minute. The rates were
obtained from regression lines fitted to the fluorescence traces over a
one minute period before and after application of the cAMP analog (see
Fig. 3A, dotted lines). As shown in Figs. 3A and 3B, 8-CPT-cAMP, activating both PKA and Epac, augmented InsP3-induced Ca2+ release significantly and in a dose-dependent manner.
Figure 3. InsP3-induced Ca2+ release is augmented by PKA activators and not by Epac activators. (A, C, D) Representative original recordings showing the effects of three cAMP analogs on InsP3-induced Ca2+ release as recorded by intraluminal Ca2+ measurements with Mag-Fura-2 in β-escin-permeabilized glands. (B, D, F) Summary of results obtained from experiments as illustrated in A, C and D. Ca2+ release is displayed as the change in the rate of the Mag-Fura-2 fluorescence ratio (ΔF340/F380·min-1)
before and after addition of a cAMP analog as shown in (A), dotted
lines. (A, B) The PKA and Epac activator 8-CPT-cAMP augments InsP3-induced Ca2+ release
significantly in a concentration-dependent manner. (C, E) Neither
8-pMeOPT-2'-O-Me-cAMP nor the two other Epac activators
(8-pHPT-2'-O-Me-cAMP and 8-pCPT-2'-O-Me-cAMP) has an effect on InsP3-induced Ca2+ release.
8-pCPT-2'-O-Me-cAMP was also ineffective in GTP-containing ICM (lowest
two bars). (D, F) All three tested PKA activators (6-Phe-cAMP,
6-BNZ-cAMP, 6-MBC-cAMP) augment InsP3-induced Ca2+ release in a concentration-dependent manner. (B, E, F) Means ± S.D., paired t-test, *P < 0.05, **P < 0.01, ***P < 0.001.
Figures 3C–F summarize the effect of three Epac-specific cAMP-analogs and of three PKA-specific analogs on InsP3-induced Ca2+ release. At a concentration of 10 μM none of the Epac activators augmented InsP3-induced Ca2+ release (Figs. 3C, E). The Epac-activator 8-pHPT-2'-O-Me-cAMP produced a slight but significant increase in the rate of Ca2+ release when applied at a concentration of 100 μM, whereas the other two Epac activators were ineffective at 100 μM. Since Epac links cAMP to the activation of the small G protein Rap1 [9,37]
and since our ICM did not contain GTP, we tested whether the above Epac
activators were ineffective because of the lack of GTP. However,
8-CPT-O-2'-Me-cAMP had also no significant effect on InsP3-induced Ca2+ release when applied in ICM supplemented with 3 mM GTP (Fig. 3E).
In contrast to the Epac activators all tested PKA-specific cAMP analogs augmented InsP3-induced Ca2+release significantly in a dose-dependent manner (Figs. 3E, F). These findings indicated that the cAMP-dependent augmentation of InsP3-induced Ca2+ release was mediated by PKA rather than Epac.
PKA inhibitors block the augmentation of InsP3-induced Ca2+ release by cAMP
To examine by an alternative approach whether the cAMP evoked augmentation of the InsP3-induced Ca2+ release was mediated by PKA, we tested the effect of the competitive antagonist of cAMP-binding to PKA, 8-Br-Rp-cAMPS [39,40], and of the PKA inhibitor H-89  on 8-CPT-cAMP-augmented InsP3-induced Ca2+ release. Both substances reversed the extra-Ca2+ release produced by 8-CPT-cAMP on a background of 5 μM InsP3 (Figs. 4A–D). These results provided further support for our conclusion that the cAMP-evoked augmentation of InsP3-induced Ca2+ release was mediated by PKA.
Figure 4. The
competitive antagonist of cAMP-binding to PKA, 8-Br-Rp-cAMPS (A, B),
and the PKA inhibitor H-89 (C, D) reverse augmentation of InsP3-induced Ca2+ release caused by 8-CPT-cAMP. Graphs constructed as described for Fig. 3.
Does cAMP-mediated augmentation of InsP3-induced Ca2+ release affect transepithelial electrolyte transport?
The transepithelial potential (TEP) is a sensitive indicator of the transepithelial K+ and Cl- transport that results from 5-HT-induced activation of the InsP3/Ca2+ and cAMP signaling pathways, because K+ transport is activated by cAMP and Cl- transport is activated by Ca2+ [34,38]. We used TEP measurements in order to examine whether cAMP was able to amplify transepithelial Cl- transport
induced (1) by 5-HT concentrations that were just sufficient to
stimulate fluid secretion and (2) by saturating 5-HT concentrations.
Because cAMP also stimulates transepithelial K+ transport by activating an apical vacuolar-type H+-ATPase that energizes K+ transport [33,42,43], we had to minimize the contribution of transepithelial K+ transport to 5-HT-induced TEP changes. This was accomplished by using a K+-free PS containing 7.5 mM of the K+ channel blocker Ba2+ to block basolateral K+ entry , as illustrated in Fig. 5A.
A brief control stimulation with 30 nM 5-HT produced a biphasic change
of the TEP. The negative-going phase of the TEP change was attributable
to transepithelial Cl- transport, and the positive-going phase was caused by the somewhat delayed transepithelial K+ transport . Superfusion of the preparation with BaCl2-containig
PS caused the TEP to become negative by about 10 mV, because the
resting TEP was slightly positive attributable to some transepithelial K+ transport in the unstimulated gland. Upon application of 1 nM 5-HT to the BaCl2-containing PS, the TEP became more negative (Fig. 5A), as a result of 5-HT-induced Ca2+ release  and a Ca2+-induced activation of transepithelial Cl- transport. Most significantly, 500 μM IBMX caused the TEP to become even more negative in the presence of 1 nM 5-HT. The effects of IBMX, 5-HT, and Ba2+ were reversible. Fig. 5B
summarizes the results of several experiments of this kind and displays
the TEP recorded at four selected time points indicated in Fig. 5A. The experiment illustrated in Fig. 5C
is identical, except that the preparation was stimulated with 30 nM
5-HT, a concentration that saturates the rate of fluid transport. At
this high 5-HT concentration, IBMX caused no further change of the TEP
Figure 5. Effects of IBMX on 5-HT-induced changes in transepithelial potential (TEP) in Ba2+-containing PS.
(A, B) Original recordings. The bar graphs (B, D) display and summarize
the TEPs recorded at the time points (1–4) as indicated in A and C;
means ± S.D. In both groups of experiments (A, C), an initial control
stimulation with 30 nM 5-HT produces a biphasic TEP change. The TEP
goes negative after superfusion of the preparation with Ba2+-containing
PS. Addition of 1 nM and 30 nM 5-HT cause the TEP to go further
negative. The TEP recorded in the presence of 1 nM 5-HT (A, B) but not
30 nM 5-HT (C, D) goes further negative by application of 500 μM IBMX in the presence of 5-HT.
The results of these TEP measurements indicate that an
increase in intracellular cAMP concentration (by application of the
phosphodiesterase inhibitor IBMX) augments the effect of a threshold
concentration of 5-HT on transepithelial Cl- transport. This result is in agreement with above finding that cAMP sensitizes the InsP3R Ca2+ channel for InsP3. The physiological consequence of InsP3R sensitization is measurable only when the glands are stimulated by low 5-HT concentrations.